The overall goal of this project is to develop conventional and conditional mouse models for junctional epidermolysis bullosa (JEB) by disrupting or affecting the expression of the gamma2 chain gene (LAMC2) of laminin 5 using Cre/loxP and FLP/frt targeting systems. These mice are used as tools towards development of therapy for JEB. Towards the overall goal of this project, the following specific aims are proposed: 1) Isolation and characterization of LAMC2 lambda-clones from mouse genomic library; 2) Construction of the targeting vector based on the data obtained from 1); 3) Generation of three allelic mouse models with different JEB phenotypes by targeting the LAMC2 gene; 4) Development of ex vivo and in vivo gene therapy protocols. Junctional epidermolysis (JEB) bullosa is a recessively inherited form of EB characterized by cutaneous and extracutaneous manifestations. There are no specific therapies available for any type of EB thus far. To achieve the specific aims of this proposal, the genomic LAMC2 clones will be isolated and characterized. This information provides basis for construction of targeting vector, which is then used for generation of three allelic mouse models utilizing Cre/loxP and FLP/frt systems. One of the models will be a conditional, tissue-specific knockout for frameshift deletion of exon 8. In this mouse the deletion of exon 8 is activated by Cre expression driven under K5 promoter. The phenotype of this mouse is expected to be milder than that of the corresponding conventional knockout mouse with the same deletion which will be developed as the second model of JEB. The third mouse model is created by FLP/frt system to target in-frame deletion of exon9. These mouse models will be used in this proposal for development of technology towards targeted correction of the mutation. For this purpose, a specific targeting vector containing the mutant region, or BAC and P1 clones containing full-length LAMC2 gene will be delivered by electroporation together with FLP protein into the cultured keratinocytes, or directly onto the skin of affected mouse. These genomic clones, fitted with frt sites, are expected to insert into the frt site present in the mouse genome created during manipulation of the mouse. The engineered cells, grafted back to the skin of the mouse, are expected to restore LAMC2 and laminin 5 expression as well as improve the skin phenotype. The development of these mouse models provides information on the different tissue involvement in the pathogenesis and the severity of the JEB. These mice will serve as tools for development of novel gene therapy protocols for JEB in future.